Cardioplegic solution for arresting an organ

ABSTRACT

There is provided cardioplegic solutions for arresting an organ intended for transplantation and preservation solutions for perfusing and storing an organ while awaiting transplantation. The cardioplegic solutions include, per liter of solution, a balanced isotonic solution of sodium, potassium, calcium, and magnesium ions and bicarbonate in a physiologically acceptable amount, from about 0.5 μM to about 2.0 μM of an amiloride-containing compound; and water sufficient to make a liter of solution. The amiloride-containing compound may be amiloride, hexamethylene amiloride, dimethyl amiloride, ethyl isopropyl amiloride or methyl isobutyl amiloride. The preserving solutions are based on the isotonic solutions described and include other components such as EDTA, a small amount of adenosine, and at least one antioxidant. There is also provided a method for arresting an organ, storing an organ and transplanting an organ all at room temperature for up to at least 24 hours.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to cardioplegic solutions, to preservationsolutions, and to methods for transplanting organs. More particularly,this invention relates to cardioplegic solutions for arresting an organfor transplantation, to preservation solutions for perfusing and storingan organ while awaiting implantation, and to methods for using thecardioplegic and preserving solutions during transplantation of anorgan.

2. The Prior Art

A great deal of research progress has been made over the years inunderstanding cellular mechanisms, as well as developing newtransplantation techniques for keeping organs viable not only duringstorage but also after reperfusion of these organs. As a result, organtransplantation, including heart transplantation, is an establishedelective operation. A significant factor limiting the clinicalapplication of organ transplantation is the deviation of viability ofthe organ after removal from the donor.

At present, the two most frequently used methods for hearttransplantation are simple hypothermic storage and continuous pulsatileperfusion. With simple hypothermic storage, the heart is arrested with acardioplegic solution, then removed from the donor and cooled rapidly.This is usually achieved by a combination of cooling and a short periodof perfusion to drop the heart temperature as quickly as possible to atemperature between 0° C. and 4° C. where it may be held up to about 6hours. While cold storage enables organs to be transplanted, the timeduring which the organ is viable is short. Cold storage decreases therate at which intracellular enzymes, essential cellular componentsnecessary for organ viability, degrade but does not stop metabolism.

The second method of organ preservation which has undergone extensiveinvestigation, continuous pulsatile perfusion, includes the followingsteps: (1) pulsatile flow, (2) hypothermia, (3) membrane oxygenation,and (4) a perfusate containing both albumin and lipids. Although beingmore technically complex and costly, the advantages to using continuouspulsatile perfusion over simple hypothermia include longer viability ofthe organ and viability testing prior to implantation.

The compositions of numerous cardioplegic and preservation solutionshave been extensively studied. For example, the protective properties ofthree cardioplegic solutions were compared by Gali nanes et al. (M. Galinanes, T. Murashita and D. J. Hearse (1991) The Journal of Heart andLung Transplantation (11) 624-635, at low temperatures and short timeperiods. A comparison of cold preservation solutions was set forth in G.Tian et al. (1991) The Journal of Heart and Lung Transplantation (10)975-985, where the cold preservation solutions limited the storage timeof the organs.

A storage solution for preserving organs which can be used attemperatures from 0° C. to 37° C. but was limited in storage time wasdisclosed in U.S. Pat. No. 5,145,771 to Lemasters et al. The solutionrequires the use of the colloid, hydroxyethyl starch, for oncoticsupport against interstitial edema. In the present invention edema isnot a problem because no oxygen-derived free radicals are available toinjure the organ.

Preserving organs at between 0° C. and 4° C. results in damage to theorgan during storage and upon reperfusion with a warm reperfusionsolution. Injury to the organ occurs through the loss of endothelialcells due to dissolved oxygen in the reperfusion solution. Although someof the solutions of the prior art have been useful to extend the storagetime of donor organs and lessen injury to the organ upon reperfusion,cell injury still occurs. Therefore, it is desirable to extend theviable organ life and improve the quality of the transplanted organ.Extending the organ viability allows sufficient time for compatibilitytesting of the donor and recipient and increased organ availability.

The recovery of cardiac function is also greatly influenced by the timelapse between removal from the donor and reperfusion as well as theefficacy of protective interventions used during that period. Toovercome the deleterious effects of ischemia, techniques such asintermittent or continuous perfusion have been used. Finally,reperfusion itself, although necessary for the survival of the tissue,may initiate a series of events known as reperfusion induced injury,which, if occurring, limit the extent or rate of recovery, Thus,modification of the nature of reperfusion is desirable to improve therecovery of the ischemic/reperfused myocardium.

More particularly, as a result of the deprivation of circulation, andthus oxygen (i.e., ischemia) during transplantation, the sodium pump,which normally maintains the intracellular composition high inpotassium, magnesium, and phosphate and low in sodium and chloride,ceases to function due to the lack of energy, resulting in an inflow ofsodium and chloride into the cells, and an outflow of potassium and to alesser extent magnesium from the cells. The result of these rapidchanges in Na⁺ --H⁺ distribution in the cell is a net gain, not merelyan exchange, of intracellular ions followed by water and a profound lossof potassium and magnesium resulting in damage to the organ. Theprotective effects of Na/H exchange inhibitors, including amiloride andits analogs in the reperfused myocardium has been studied by Moffat, etal. (M. P. Moffat and M. Karmazyn, (1993), J. Molec. Cell Cardiol (25),959-971).

It is therefore the general object of the present invention to providecardioplegic solutions for arresting organs intended for transplantationand preserving solutions for pulsating and storing organs while awaitingimplantation each of which inhibits ion exchange, extends the vitalityof the organ, and reduces damage to the cells.

Another object of the present invention is to provide a method forarresting and preserving organs which extend the maximum life of theorgan during transplantation.

Yet another object of the present invention is to provide a method oftransplanting organs in which storage of the organ may be carried out atroom temperature for up to at least 24 hours without significant damageto the organ.

Other objects, features, and advantages of the invention will beapparent from the following details of the invention as more fullydescribed.

SUMMARY OF THE INVENTION

In accordance with these objects and the principles of this invention,there are disclosed cardioplegic solutions and preserving solutions foruse in the transplantation of organs, and to methods for transplantingorgans using the solutions, which methods increase storage times andlessen injury to the organ.

It has been found that a cardioplegic solution containing anamiloride-containing compound is effective in achieving the objectivesof this invention. In one aspect of this invention, the cardioplegicsolution includes a balanced isotonic solution including sodium,potassium, calcium and magnesium ions and bicarbonate in aphysiologically acceptable amount, from about 0.5 μM to about 2.0 μMamiloride-containing compound, and water sufficient to make a liter ofsolution.

The inclusion in the isotonic solution of an amiloride-containingcompound may be amiloride itself, or amiloride analogs, such ashexamethylene amiloride (HMA), dimethyl amiloride (DMA), ethyl isopropylamiloride (EIPA), or methyl isobutyl amiloride (MIA), all of whichinhibit the Na⁺ --H⁺ exchange in the organ cells. Dimethyl amiloride isparticularly preferred.

The cardioplegic solution also preferably contains glucose to enhanceorgan preservation, adenosine to prevent fibrillation of the organ priorto removal from the donor, and EDTA as a chelating agent. Optionally,the cardioplegic solution contains heparin and at least one antioxidant.

The preservation solution, while similar to the cardioplegic solution instarting composition in that it is based on a balanced isotonic solutionincluding sodium, potassium, calcium, magnesium ions and bicarbonate ina physiologically acceptable amount differs in significant respects. Forexample, the preservation solution includes less potassium chloride thanthe cardioplegic solution and no amiloride. In addition, since the organhas been arrested by the cardioplegic solution, when the preservationsolution includes much less adenosine and heparin is not normallyneeded.

The preservation solution preferably includes at least one antioxidant,such as, dimethyl thiourea (DMTU), catalase as a hydrogen peroxidescavenger and apoferritin to decrease iron content within thepreservation solution. In addition, the preservation solutionsoptionally may include hormones, such as insulin and prostaglandin andantibiotics.

The invention also provides a method for transplanting an organ whichincludes steps for arresting and removing the organ from the donor, andfor preserving and storing the organ intended for implantation. Themethod of the invention includes arresting the organ to be donated withthe cardioplegic solution. The organ is removed and connected to aperfusion apparatus where it is maintained at a temperature betweenabout 0° C. to about 37° C., preferably from about 15° C. to about 25°C. while perfusing with the preservation solution. Thus, the novelfeatures of the present invention include storing the organ at warmtemperatures, i.e., up to about room temperature, while perfusing theorgan. It is believed that the ability to store the organ at or nearroom temperature prevents mechanical damage that can result from coldstorage, and that continuous perfusion with the preservation solutionmaintains the organ's metabolic requirements and avoids potentialmetabolic blocks. As a result, storage times for the organ can beincreased up to at least 24 hours.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to new cardioplegic solutions for anarresting organ intended for transplantation and to new preservationsolutions for storing and perfusing organs intended for implantation ina patient requiring such implant. Suitable organs on which the solutionsof this invention may be used include, for example, heart, liver, kidneyand pancreas.

The individual components of the present invention are all nontoxic andhave been found to be stable during storage. While some of thecomponents of the present invention are similar to those of other knowncardioplegic solutions, it has surprisingly been found that the additionof amiloride or an amiloride-containing compound to a balanced isotonicsolution including sodium, potassium, calcium, magnesium, andbicarbonate ions in a physiologically acceptable amount to form acardioplegic solution and its use in combination with the preservationsolutions of the present invention allows organs to be preserved at roomtemperature for at least 24 hours without significant damage to theorgan.

Both the cardioplegic solutions and the preservation solutions of thepresent invention are based on a balanced isotonic solution includingsodium, potassium, calcium and magnesium ions as well as glucose andsodium bicarbonate in a physiologically acceptable amount. Certain ofthese types of solutions are well known, such as the one describedbelow, known as Krebs-Henseleit-bicarbonate solution, which has thefollowing composition:

                  TABLE 1                                                         ______________________________________                                        Concentration Ranges in 1 Liter                                               ______________________________________                                        NaCl         85 mM to 145 mM                                                  KCl          3 mM to 30 mM                                                    CaCl.sub.2  0.5 mM to 2.5 mM                                                  KH.sub.2 PO.sub.4                                                                         0.7 mM to 1.3 mM                                                  MgSO.sub.4  0.9 mM to 4.8 mM                                                  NaHCO.sub.3 15 mM to 35 mM                                                    Glucose     1.0 mM to 50 mM                                                   ______________________________________                                    

The Cardioplegic Solution

The cardioplegic solution is made by starting with the balanced isotonicsolution described above. The amount of potassium chloride in thecardioplegic solution is preferably from about 20 mM to about 30 mM. Tothe balanced isotonic solution is added an amiloride-containing compoundin an amount from about 0.5 μM to about 2.0 μM, preferably 1.0 μM. Byamiloride-containing compound, it is meant to include amiloride andamiloride analogs. The addition of amiloride, designated chemically as3,5-diamino-6-chlor-N-(diaminomethylene) pyrazinecarboxamidemonohydrochloride has been found to inhibit the Na⁺ --H⁺ exchange. Otheramiloride-containing compounds or analogues which may be used in thecardioplegic solution include, for example, hexamethylene amiloride(HMA) designated chemically as 5-(N,N-hexamethylene)amiloride, dimethylamiloride (DMA) designated 5-(N,N-dimethyl)-amiloride, ethyl isopropylamiloride (EIPA), designated 5-(N-ethyl-N-isopropyl)-amiloride, andmethyl isobutyl amiloride (MIA), designated5-(N-methyl-N-isobutyl)-amiloride. (Merck Sharpe & Dohme, West Point,Pa.).

To be effective, the cardioplegic solution needs to prevent fibrillationof the organ in a relatively short period of time, e.g., 2 minutes to 5minutes or less. For that purpose, adenosine is added to the solution inan amount from about 5 μM to about 15 μM per liter, preferably about 10μM per liter. Adenosine rapidly arrests the heart (within seconds),improves the preservation properties and increases the glucose uptake.

The cardioplegic solution also preferably includes ethylenediaminetetraacetic acid (EDTA) in an amount from 0.5 mM to about 1.5 mMas a chelating agent. The solution also may optionally contain otheringredients, such as at least one antioxidant, for example, catalase, inan amount effective to inhibit the generation of oxygen-derived freeradicals via hydrogen peroxide.

Heparin is used in carrying out the method of this invention, and may beincluded either directly in the cardioplegic solution or it may beadministered to the donor organ separately. The addition of heparin inan amount of from about 500 units to about 1500 units, preferably 1000units is required to prevent blood clots from forming within thecoronary arteries during cardioplegic arrest and excision prior to organstorage and implantation.

The Preservation Solution

The preservation solution is designed to prevent various mechanismswhich cause injury to the organ and thus must be a composition that (1)prevents or restricts intracellular acidosis, (2) prevents the expansionof intracellular space, (3) prevents injury from oxygen-derived freeradicals, especially during reperfusion, (4) enables the regeneration ofhigh-energy phosphate compounds during reperfusion, and (5) sustainsappropriate metabolic requirement.

While the cardioplegic solution and the preservation solution begin withthe balanced isotonic solution described above there are significantdifferences in the final compositions. For example, the preservationsolution begins with the isotonic solution, wherein the potassiumconcentration is maintained at preferably from 3 mM to about 8 mM.Magnesium chloride may be used in place of potassium chloride.

While adenosine is included in the preservation solution, the amount ofadenosine is considerably less than the amount of adenosine present inthe cardioplegic solution because the organ has been previouslyarrested. The amount of adenosine in the preservation solution isnormally from 0.7 μM to about 2.0 μM, preferably about 1.0 μM.

The cardioplegic solution also preferably includes ethylenediaminetetraacetic acid (EDTA) in an amount from 0.5 mM to about 1.5 mMas a chelating agent. It has also been found desirable to add from 10 nMto about 100 nM of caprylic acid which helps the solution to bypassblocked fatty acid utilization and from 10 μg/L to 100 μg/L ofapoferritin which serves to eliminate iron (Fe++) which causes breakdownof the cells.

Suitable antioxidants include, but are not limited to, allopurinol,desferrioxamine, glutathione, beta-carotene, catalase, superoxidedismutase, dimethyl thiourea (DMTU), dichloroacetic acid, diphenylphenylene diamine (DPPD), mannitol or cyanidanol in an amount effectiveto inhibit the generation of oxygen-derived free radicals. Theantioxidants are present in an amount from 1 nM to 10 nM.

Optional components ingredients include, for example, hormones, such asinsulin and prostaglandin and antibiotics.

Method of Use

The transplantation method of the present invention is to arrest theorgan using the cardioplegic solution, preserve and store the organ withthe preservation solution and reperfuse with the preservation solution.

In a preferred method, sufficient cardioplegic solution is injected toarrest, for example the heart and prevent fibrillation. The surgeon thenremoves the organ and connects the heart to a perfusion apparatuscomprising tubing and pumps. The preservation solution is then perfusedthrough the heart while gassed with oxygen and carbon dioxide while itis awaiting implantation into a patient. The method of perfusing theheart can be at either a constant flow or pressure.

The solution can be used at all temperatures ranging from 0° C. tonormal body temperature, 37° C. At temperatures of from about 12° C. toabout 37° C., the solution is more protective than other knownpreservation solutions. Unlike other storage solutions, it continues tobe protective above 10° C. for at least 24 hours.

The following examples are provided to further illustrate the presentinvention and are not to be construed as limiting the invention in anymanner.

EXAMPLE 1

A liter of cardioplegic solution having the following composition wasprepared.

    ______________________________________                                        NaCl                   118    mM                                              KCl                    30     mM                                              CaCl.sub.2             1.75   mM                                              KH.sub.2 PO.sub.4      1.2    mM                                              MgSO.sub.4             1.2    mM                                              NaHCO.sub.3            25     mM                                              Glucose                11     mM                                              Adenosine              10     μM                                           EDTA                   1.0    mM                                              DMA                    1.0    μM                                           Heparin                1000   units                                           Distilled, deionized water                                                                           q.s.                                                   ______________________________________                                    

Into a 1000 mL volumetric flask the Krebs-Henseleit solution was addedand double distilled water was added to make one liter while stirring.The rest of the components were added one at a time. After all thecomponents were added the pH was adjusted to 7.3 with NaOH and the flaskwas gassed with O₂ /CO₂ :95/5%. The solution was stirred for aboutthirty minutes and filtered to remove any undissolved particles (5.0μporosity filter). After sterile filtration the solution was ready touse.

EXAMPLE 2

A liter of preservation solution having the following composition wasprepared.

    ______________________________________                                        NaCl                  118    mM                                               KCl                   4.7    mM                                               CaCl.sub.2            1.75   mM                                               KH.sub.2 PO.sub.4     1.2    mM                                               MgSO.sub.4            1.2    mM                                               NaHCO.sub.3           25     mM                                               Glucose               11     mM                                               Adenosine             1.0    μM                                            EDTA                  1.0    mM                                               Apoferritin           100    μg/L                                          Catalase              10     μg/L                                          DMTU                  10     nM                                               DPPD                  10     nM                                               Caprylic Acid         50     nM                                               Distilled, deionized water                                                                          q.s.                                                    ______________________________________                                    

The preservation solution was prepared in much the same manner as thecardioplegic solution of Example I, that is, by adding aKrebs-Heneseleit solution to a 1000 mL volumetric flask with doubledistilled water to make one liter while stirring. The rest of thecomponents were added one at a time and the pH of the solution wasadjusted to about 7.3 with NaOH and gassed with 95% oxygen plus 5%carbon dioxide. The solution was stirred for about thirty minutes andfiltered to remove any undissolved particles (5.0μ porosity filter).After sterile filtration the solution was ready to use.

EXAMPLE 3

A female mongrel dog weighing about 20 kg. was anesthetized. An IVhydrating solution of 5.0% dextrose in 0.45% saline at 75 cc/hr wasgiven throughout the procedure. The heart was exposed by a sternotomy.The cardioplegic solution of Example 1 was administered to arrest theheart, the heart was then excised. The heart was placed in ice andpromptly transferred to the laboratory and placed in a perfusionapparatus at room temperature where the aorta was attached to a tube forcontinuous perfusion with the preserving solution of Example 2. After2-3 minutes the heart started beating at a pulse rate of 50 beats perminute. Excess preserving solution was allowed to fill the containeruntil the heart was covered to provide buoyancy for the heart so as notto injure the aorta. Perfusion with the solution from Example 2continued throughout the storage time. The heart continued to beat forover 24 hours.

This experiment demonstrates that the cardioplegic and preservationsolutions and methods of the present invention increase the preservationtime between harvesting an organ and transplantation and allows theorgan to remain at room temperature without serious degradation of theorgan cells.

The present invention has been described in detail and with particularreference to the preferred embodiments. Those skilled in the art willappreciate that changes can be made without departing from the spiritand scope thereof. Accordingly, the present invention is to be definedby the following claims, with equivalents of the claims to be includedtherein.

What is claimed is:
 1. A cardioplegic solution for arresting a heartintended for transplantation comprising, per liter of solution:(a) abalanced isotonic solution comprising sodium, potassium, calcium,magnesium and bicarbonate ions in a physiologically acceptable amount;(b) from about 0.5 μM to about 2.0 μM of an amiloride-containingcompound; and (c) water.
 2. The cardioplegic solution according to claim1, wherein said amiloride-containing compound is a compound selectedfrom the group consisting of amiloride, hexamethylene amiloride,dimethyl amiloride, ethyl isopropyl amiloride and methyl isobutylamiloride.
 3. The cardioplegic solution according to claim 1, whereinsaid amiloride-containing compound is dimethyl amiloride.
 4. Thecardioplegic solution according to claim 1, further comprising fromabout 0.5 mM to about 1.5 mM of EDTA.
 5. The cardioplegic solutionaccording to claim 1, further comprising from about 5.0 μM to about 15μM of adenosine.
 6. The cardioptegic solution according to claim 1,further comprising from about 500 units to about 1500 units of heparin.7. The cardioplegic solution according to claim 1, further comprising anantioxidant in an amount effective to inhibit the generation ofoxygen-derived free radicals.
 8. A cardioplegic solution for arresting aheart intended for transplantation comprising, per liter of solution:

    ______________________________________                                        NaCl               85 mM to 145 mM                                            KCl               20 mM to 30 mM                                              CaCl.sub.2        0.5 mM to 2.5 mM                                            KH.sub.2 PO.sub.4 0.7 mM to 1.3 mM                                            MgSO.sub.4        0.9 mM to 4.8 mM                                            NaHCO.sub.3       15 mM to 35 mM                                              Glucose           1.0 mM to 50 mM                                             EDTA              0.5 mM to 1.5 mm                                            Distilled, deionized water                                                                      q.s.                                                        ______________________________________                                    

and from about 0.5 μM to about 2.0 μM of an amiloride-containingcompound.
 9. The cardioplegic solution according to claim 8, whereinsaid amiloride-containing compound is a compound selected from the groupconsisting of amiloride, hexamethylene amiloride, dimethyl amiloride,ethyl isopropyl amiloride and methyl isobutyl amiloride.
 10. Thecardioplegic solution according to claim 8, wherein said amiloridecontaining compound is dimethyl amiloride.
 11. The cardioplegic solutionaccording to claim 8, further comprising from about 5.0 μM to about 15μM of adenosine.
 12. The cardioplegic solution according to claim 8,further comprising from about 500 units to about 1500 units of heparin.13. The cardioplegic solution according to claim 8, further comprisingan antioxidant in an amount effective to inhibit the generation ofoxygen-derived free radicals.
 14. A cardioplegic solution for arrestinga heart intended for transplantation comprising, per liter of solution:

    ______________________________________                                        NaCl                   118    mM                                              KCl                    30     mM                                              CaCl.sub.2             1.75   mM                                              KH.sub.2 PO.sub.4      1.2    mM                                              MgSO.sub.4             1.2    mM                                              NaHCO.sub.3            25     mM                                              Glucose                11     mM                                              Adenosine              10     μM                                           EDTA                   1.0    mM                                              Dimethyl amiloride     1.0    μM                                           Heparin                1000   units                                           Distilled, deionized water                                                                           q.s.                                                   ______________________________________                                    


15. A method for removing a heart from a mammal intended forimplantation in a mammal requiring such implantation, said methodcomprising:arresting said heart prior to removal from a donor with asolution comprising, per liter of solution: (a) a balanced isotonicsolution comprising sodium, potassium, calcium, magnesium, andbicarbonate ions in a physiologically acceptable amount; (b) from about0.5 μM to about 2.0 μM of an amiloride-containing compound; and (c)water.
 16. The method according to claim 15, in which saidamiloride-containing compound is a compound selected from the groupconsisting of amiloride, hexamethylene amiloride, dimethyl amiloride,ethyl isopropyl amiloride and methyl isobutyl amiloride.
 17. The methodaccording to claim 15, in which said amiloride-containing compound isdimethyl amiloride.
 18. The method according to claim 15, in which saidsolution further comprises from about 0.5 mM to about 1.5 mM EDTA. 19.The method according to claim 15, in which said solution furthercomprising from about 5.0 μM to about 15 μM of adenosine.
 20. The methodaccording to claim 15, in which said solution further comprising fromabout 500 units to about 1500 units of heparin.
 21. The method accordingto claim 15, in which said solution further comprising at least oneantioxidant in an amount effective to inhibit the generation ofoxygen-derived free radicals.
 22. A method for removing the heart from amammal intended for transplantation, said method comprising:arrestingthe heart prior to removal from a donor with a solution comprising, perliter of solution:

    ______________________________________                                        NaCl               85 mM to 145 mM                                            KCl                3 mM to 30 mM                                              CaCl.sub.2        0.5 mM to 2.5 mM                                            KH.sub.2 PO.sub.4 0.7 mM to 1.3 mM                                            MgSO.sub.4        0.9 mM to 4.8 mM                                            NaHCO.sub.3       15 mM to 35 mM                                              Glucose           1.0 mM to 50 mM                                             EDTA              0.5 mM to 1.5 mm                                            Distilled, deionized water                                                                      q.s.                                                        ______________________________________                                    

and from about 0.5 μM to about 2.0 μM of an amiloride-containingcompound.
 23. The method according to claim 22, wherein saidamiloride-containing compound is a compound selected from the groupconsisting of amiloride, hexamethylene amiloride, dimethyl amiloride,ethyl isopropyl amiloride and methyl isobutyl amiloride.
 24. The methodaccording to claim 22, wherein said amiloride-containing compound isdimethyl amiloride.
 25. The method according to claim 22, in which saidsolution further comprising from about 5 μM to about 15 μM of adenosine.26. The method according to claim 22, in which said solution furthercomprising from about 500 units to about 1500 units of heparin.
 27. Themethod according to claim 22, in which said solution further comprisingat least one antioxidant in an amount effective to inhibit thegeneration of oxygen-derived free radicals.
 28. A method for removing aheart from a mammal intended for implantation, said methodcomprising:arresting the heart prior to removal from a donor with asolution comprising, per liter of solution:

    ______________________________________                                        NaCl                   118    mM                                              KCl                    30     mM                                              CaCl.sub.2             1.75   mM                                              KH.sub.2 PO.sub.4      1.2    mM                                              MgSO.sub.4             1.2    mM                                              NaHCO.sub.3            25     mM                                              Glucose                11     mM                                              Adenosine              10     μM                                           EDTA                   1.0    mM                                              Dimethyl amiloride     1.0    μM                                           Heparin                1000   units                                           Distilled, deionized water                                                                           q.s.                                                   ______________________________________                                    